Method for preparing highly branched starch by using double enzymes

Abstract

The invention discloses a method for preparing highly branched starch by using double enzymes, and belongs to the field of biologically modified starch. According to the invention, glycogen branching enzyme and cyclodextrin glucosyltransferase are adopted to synergistically modify different crystal forms of starch in a non-gelatinization swelling system (60 DEG C) to prepare highly branched starch, the reaction is carried out on the premise of ensuring the morphology of starch particles, and the later processing application characteristics are ensured, so that the highly branched starch can be fused with a traditional starch staple food carrier, and the preparation process is simple and convenient. According to the method, the enzyme modified starch and the swelling reaction are synchronously carried out, the step of cooling after high-temperature gelatinization is reduced, the production efficiency is improved, the adverse effect of high viscosity caused by high-temperature gelatinization on starch products is avoided, and the obtained product is high in yield, high in branching degree, low in digestibility and high in glucosidic bond proportion; a healthy and green new thought is provided for the field of starch deep processing.

Description

Technical Field

[0001] The invention relates to a method for preparing highly branched starch by utilizing double enzymes, and belongs to the field of biologically modified starch. Background Art

[0002] Starch is the most important carbohydrate for humans and the main source of energy for the human body. Natural starch includes type A, type B and type C. However, natural starch is insoluble in cold water, easily ages and shrinks, and can cause a large fluctuation in blood sugar index within a short period of time after ingestion. Enzyme-modified starch can replace fat in some food industries and can also protect the transportation of some sensitive ingredients and bioactive substances.

[0003] The modified branched starch has more branching degree than natural starch, which can significantly improve the branching degree, amylose content, retrogradation, and digestion characteristics of the original starch. This is of great significance for the development of low GI foods that are friendly to obese or diabetic patients. However, the effect of single enzyme modification of starch is limited, so there is currently a method of obtaining branched starch by modifying starch with two enzymes. For example, the article "Pre-treatment of granular rice starch to enhance branching enzyme catalysis" discloses a method for preparing branched starch using two enzymes in a gelatinized state, mainly by treating rice starch with α-amylase and glycogen branching enzyme, but the obtained branched starch still has problems such as low branching degree and low glycosidic bond ratio, which still limits the application of starch in the food industry. In addition, in order to achieve an effective bond conversion rate, the focus of enzymatic modified starch at home and abroad is carried out under gelatinization conditions, which destroys the granular structure of starch and damages its unique processing properties, such as gelatinization properties, gel properties and rheological properties. It can only be consumed by dissolving or dispersing in beverages and paste foods, and cannot be integrated with traditional starch staple food carriers. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for preparing highly branched starch, which mainly adopts glycogen branching enzyme and cyclodextrin glucosyltransferase to synergistically modify different crystalline starches in a non-gelatinized swelling system (60°C) to prepare highly branched starch. The present invention performs enzymatic hydrolysis reaction based on the non-gelatinized swelling state of starch, realizes the simultaneous heating swelling and enzymatic hydrolysis reaction of starch, maintains a relatively complete starch granule structure, ensures its processing and application characteristics such as later gelatinization, reduces reaction energy consumption, avoids the problem caused by high viscosity of starch after gelatinization, shortens the processing steps required for cooling after gelatinization, and obtains a product with high yield, high branching degree, low digestibility, high glycosidic bond ratio, and low RDS content.

[0005] The present invention provides a method for preparing branched starch, which mainly comprises the following steps:

[0006] (1) dispersing starch in a buffer to obtain starch milk, and preheating the milk at 50-60° C.;

[0007] (2) adding cyclodextrin glucosyltransferase and glycogen branching enzyme to carry out enzymatic hydrolysis reaction;

[0008] (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated, the starch is centrifuged, washed, dried, ground and sieved to obtain branched starch.

[0009] In one embodiment of the present invention, in step (1), the mass concentration of starch milk is 10-20%; and the buffer solution is 0.05 mol / L sodium citrate buffer solution.

[0010] In one embodiment of the present invention, in step (1), the starch is a starch having an amylose content greater than 1%, including one or more of corn starch, potato starch, and pea starch.

[0011] In one embodiment of the present invention, in step (2), the glycogen branching enzyme is from Pyrococcushorikoshii OT3 (Ph), and the cyclodextrin glucosyltransferase is from Bacillus licheniformis.

[0012] In one embodiment of the present invention, in step (2), the amount of cyclodextrin glucosyltransferase added is 0.05-0.15 mL / g dry starch.

[0013] In one embodiment of the present invention, in step (2), the amount of glycogen branching enzyme added is 100-500 U / g dry starch.

[0014] In one embodiment of the present invention, in step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added to carry out enzymatic hydrolysis reaction, which specifically includes: after adding cyclodextrin glucosyltransferase for reaction, adjusting the pH to 11-12, standing to inactivate the enzyme, then adjusting the pH to 5-7, and finally adding glycogen branching enzyme for reaction.

[0015] In one embodiment of the present invention, in step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added to carry out enzymatic hydrolysis reaction, which specifically includes: after adding glycogen branching enzyme reaction, adjusting the pH to 11-12, standing to inactivate the enzyme, then adjusting the pH to 5-7, and then adding cyclodextrin glucosyltransferase reaction.

[0016] In one embodiment of the present invention, in step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added to carry out enzymatic hydrolysis reaction, which specifically includes: adding cyclodextrin glucosyltransferase and glycogen branching enzyme at the same time to carry out reaction.

[0017] In one embodiment of the present invention, in step (3), anhydrous ethanol is used to inactivate the enzyme.

[0018] The present invention provides branched starch prepared by the method described above.

[0019] The present invention also provides application of the branched starch in the food industry.

[0020] [Beneficial Effects]

[0021] (1) The present invention utilizes a bio-enzymatic method to modify starch to prepare branched starch. Cyclodextrin glucosyltransferase is a type of α-amylase, which performs three reactions, namely cyclization, disproportionation, and coupling, with the production of cyclodextrin. Cyclodextrin is easily soluble in water and is lost by washing. Glycogen branching enzyme can specifically act on starch molecules and increase the degree of starch branching by rearranging α-1,4 glycosidic bonds and α-1,6 glycosidic bonds. This enzymatic recombination process does not introduce any foreign chemical groups, so the resulting modified starch has higher safety.

[0022] (2) The present invention is based on the reaction at 60°C, which maintains a relatively complete starch granule structure, ensures its processing and application characteristics such as later gelatinization, enables it to be integrated with traditional starch staple food carriers, and at the same time avoids the problem of high viscosity after high-temperature starch treatment, realizes starch swelling and enzymatic hydrolysis reaction at the same time, shortens the reaction steps, and improves the efficiency of industrial production. It provides an innovative green and low-carbon solution for the production of branched starch. This method not only saves energy and reduces consumption, but is also environmentally friendly and represents a new direction for sustainable development.

[0023] (3) Compared with the prior art, the present invention solves the problems of low conversion rate, long action time and low product yield of single glycogen branching enzyme. The porous starch is prepared by pretreatment with cyclodextrin glucosyltransferase, forming a pore structure of different sizes, opening up the original tight arrangement of starch chains, providing more possible substrates for glycogen branching enzyme, and reducing the steric hindrance of the action site of glycogen branching enzyme binding to the substrate, increasing the product yield (more than 75%) and the branching degree.

[0024] (4) Compared with natural starch, the branched starch obtained by the present invention has a maximum increase of 14.9% in α-1,6 bond content; a decrease of 36.14% in in vitro digestibility in the fast digestibility; and a maximum decrease of 7.59 J / g in the gelatinization enthalpy (ΔH) in thermodynamic properties; and a significant decrease in starch viscoelasticity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The starch granule morphology of Examples 1-9;

[0026] Figure 2 Viscoelastic properties of corn branched starch prepared in Examples 1, 4, and 7;

[0027] Figure 3 Viscoelastic properties of potato branched starch prepared in Examples 2, 5 and 8;

[0028] Figure 4 The viscoelastic properties of pea branched starch prepared in Examples 3, 6 and 9;

[0029] Figure 5 It is the glycosidic bond ratio of the branched starch prepared in Examples 1-9. DETAILED DESCRIPTION

[0030] In the following embodiments, cyclodextrin glucosyltransferase is derived from Bacillus licheniformis, with an enzyme activity of 3KNU-CP / g, purchased from Novozymes (China) Biotechnology Co., Ltd.; glycogen branching enzyme is derived from Pyrococcushorikoshii OT3 (Ph), and the preparation method of the enzyme is referred to the literature: Jing Zhu, Jie Long, Xingfei Li, et al. Improving the thermal stability and branching efficiency of Pyrococcushorikoshii OT3 glycogen branching enzyme [J]. International Journal of Biological Macromolecules 255 (2024) 128010. Corn starch, potato starch, and pea starch belong to type A starch, type B starch, and type C starch, respectively.

[0031] The detection methods involved in the following embodiments are as follows:

[0032] Starch granule morphology determination:

[0033] Take the enzyme-modified starch sample, fix it on the sample stage of the scanning electron microscope (SEM) with black double-sided tape, then apply a thin layer of gold film, and then place the sample stage under the scanning electron microscope to magnify and observe the particle morphology characteristics of the three crystalline starches. The acceleration voltage is 3.0 kV.

[0034] In vitro digestibility test:

[0035] Prepare 0.75mol / L HCl, take 75mL, add 5mg pepsin and 25mg guar gum to form pepsin solution, prepare and use immediately, store at 4℃. Dissolve 2g porcine pancreatic amylase in 40mL ultrapure water, stir magnetically for 20min, centrifuge at 8000rpm for 10min, and take 30mL of supernatant. Add 0.4mL saccharifying enzyme and mix thoroughly to prepare mixed enzyme solution, prepare and use immediately, and store at 4℃. Take 200mg (dry basis) sample and add 15mL sodium acetate buffer (0.2mol / L, pH 5.0), place in a 95℃ water bath, vortex gelatinize for 30min, place in a 37℃ water bath shaker (200rpm) for 15min, and add 10 glass beads. Add 2mL pepsin solution and react for 30min. Then, 1 mL of mixed enzyme solution was added, and 0.2 mL of the reaction solution was taken at 20 min and 120 min, respectively, and 5 times anhydrous ethanol was added to inactivate the enzyme. The obtained mixture was centrifuged at 9000 rpm for 5 min, and 0.1 mL of the supernatant was taken.

[0036] The specific formula is as follows:

[0037] RDS(%)=(G20-FG)×0.9 / TS

[0038] SDS (%) = (G120-G20) × 0.9 / TS

[0039] RS(%)=100-RDS(%)-SDS(%)

[0040] in:

[0041] FG-free glucose content in starch before enzymatic hydrolysis (mg);

[0042] G20-glucose content produced after 20 min of amylase hydrolysis (mg);

[0043] G120-glucose content produced after 120 min of amylase hydrolysis (mg);

[0044] TS-total starch content in the sample (mg).

[0045] Determination of thermodynamic properties:

[0046] Use differential scanning calorimetry (DSC) to measure the changes in the thermodynamic properties of modified starch. Accurately weigh 2 mg of modified starch in an aluminum crucible, add deionized water at a ratio of 1:2 (w / w), and seal the crucible immediately. Place the crucible in a sealed bag and place it in a 4°C refrigerator to balance the moisture for 12 hours. Raise the temperature from 20°C to 100°C at a heating rate of 10°C / min, and keep the nitrogen flow rate constant at 20mL / min. Use a sealed empty crucible as a control. Each sample is measured three times in parallel, and the starting temperature (T O), peak temperature (T P ), termination temperature (T C ) and enthalpy (ΔH).

[0047] Gelatinization properties determination:

[0048] Take 2g of the sample before and after modification and add 16.5mL of deionized water. Place it in an aluminum box for rapid viscosity analyzer (RVA) test. In order to prevent starch granules from settling, the starch suspension was continuously stirred manually with a matching plastic stirring paddle before the test. The specific heating procedure is as follows: the sample is first kept at 50°C for 1min, then heated to 95°C at a rate of 5.4°C / min, and kept at 95°C for 5min. Then, the temperature is cooled from 95°C to 50°C at a rate of 5.4°C / min, and kept at 50°C for 2min. Within 10s after the start of the experiment, the speed of the stirring paddle is 960rpm to fully disperse the sample; then the speed is reduced and maintained at 160rpm. The RVA instrument automatically records the changes in sample viscosity with heating time and temperature during the above-mentioned heating-constant temperature-cooling process.

[0049] Determination of α-1,6 glycosidic bond ratio:

[0050] Weigh 10 mg of modified starch sample and disperse it in 0.5 mL of D2O. Heat it in a boiling water bath to make it evenly dispersed. Vortex and shake it every 10 minutes until the heating is stopped after 1 hour. After freezing at -80℃ overnight, put it in a freeze dryer and dry it in vacuum for 2 days. When measuring, the sample is redissolved in D2O with internal standard. Use a nuclear magnetic resonance spectrometer to record the nuclear magnetic resonance ( 1 H NMR) spectra with a spectral width of 8000 Hz, and data processing was performed using Mestre Nova 12.0.3 software.

[0051] Example 1

[0052] Corn starch was dispersed in a sodium citrate buffer solution with a pH of 5.0 and a concentration of 0.05 mol / L to prepare a 10% wt starch milk, preheated at 60°C for 30 min, 0.1 mL of cyclodextrin glucosyltransferase (per g of dry starch) was added to react for 6 h, the pH was adjusted to 11.5, the enzyme was allowed to stand for 15 min, the pH was adjusted to 6.0, glycogen branching enzyme (500 U / g dry starch) was added to react for 20 h, 3 times anhydrous ethanol was added to kill the enzyme, the enzyme was allowed to stand for 30 min, centrifuged at 10000 rpm for 10 min, and then washed with deionized water for 3 times. The precipitate was frozen at -80°C for 24 h, vacuum freeze-dried, and ground through a 100-mesh sieve to obtain a highly branched starch sample, which was named CS-C / G.

[0053] Example 2

[0054] The preparation method is consistent with that in Example 1, except that corn starch is replaced by potato starch, and the prepared highly branched starch is named PS-C / G.

[0055] Example 3

[0056] The preparation method is consistent with that in Example 1, except that corn starch is replaced by pea starch, and the prepared highly branched starch is named PBS-C / G.

[0057] Example 4

[0058] Corn starch was dispersed in a sodium citrate buffer solution with a pH of 5.0 and a concentration of 0.05 mol / L to prepare a 10% wt starch milk, preheated at 60°C for 30 min, 300 U / g dry starch glycogen branching enzyme was added to react for 10 h, the pH was adjusted to 11.5, the enzyme was allowed to stand for 15 min, the pH was adjusted to 7.0, 0.05 mL (per g dry starch) of cyclodextrin glucosyltransferase was added to react for 18 h, 3 times anhydrous ethanol was added to kill the enzyme, the enzyme was allowed to stand for 30 min, centrifuged at 10000 rpm for 10 min, and then washed with deionized water for 3 times. The precipitate was frozen at -80°C for 24 h, vacuum freeze-dried, and ground through a 100-mesh sieve to obtain a highly branched starch sample, which was named CS-G / C.

[0059] Example 5

[0060] The preparation method is consistent with that in Example 4, except that corn starch is replaced with potato starch, and the prepared highly branched starch is named PS-G / C.

[0061] Example 6

[0062] The preparation method is consistent with that in Example 4, except that corn starch is replaced by pea starch, and the prepared highly branched starch is named PBS-G / C.

[0063] Example 7

[0064] Corn starch was dispersed in a sodium citrate buffer solution with a pH of 5.0 and a concentration of 0.05 mol / L to prepare a 10% wt starch milk, preheated at 60°C for 30 min, 0.05 mL of cyclodextrin glucosyltransferase (per g of dry starch) was added, 300 U / g of dry starch glycogen branching enzyme was added to react for 10 h, 3 times anhydrous ethanol was added to inactivate the enzyme, the mixture was allowed to stand for 30 min, centrifuged at 10000 rpm for 10 min, and then washed with deionized water for 3 times. The precipitate was frozen at -80°C for 24 h, vacuum freeze-dried, and ground through a 100-mesh sieve to obtain a highly branched starch sample named CS-C+G.

[0065] Example 8

[0066] The preparation method is consistent with that in Example 7, except that corn starch is replaced with potato starch, and the prepared highly branched starch is named PS-C+G.

[0067] Example 9

[0068] The preparation method is consistent with that in Example 7, except that corn starch is replaced by pea starch, and the prepared highly branched starch is named PBS-C+G.

[0069] Starch granule morphology:

[0070] from Figure 1 It can be seen that at 60°C, the double-enzyme modified corn starch, potato starch, and pea starch (Examples 1-9) all maintained complete granular morphology, and the non-gelatinization system of the present invention ensured the later processing and application characteristics of the modified starch.

[0071] Gelatinization property analysis:

[0072] Depend on Figure 2-4 It can be seen that corn starch, potato starch and pea starch all present the common RVA test curve, with the viscosity first increasing and then decreasing, and the viscosity of the product is greatly reduced compared with the viscosity of the starch before modification.

[0073] In vitro digestibility analysis:

[0074] In the in vitro simulated digestion assay, the results show (as shown in Table 1) that the proportion of fast-digestible starch content in the branched starch obtained by the dual enzyme modification of the present invention is reduced to less than 70%. After pretreatment with cyclodextrin glucosyltransferase and adding glycogen branching enzyme, the fast-digestible starch content of corn starch, potato starch, and pea starch was reduced by 36.14%, 20.03%, and 19.62% compared with the original starch, the slowly digestible starch content of corn starch increased by 20.14%, and the resistant starch content increased by 21.07%.

[0075] Table 1 RDS, SDS, RS content of Examples 1-9

[0076]

[0077] Thermodynamic property analysis:

[0078] As shown in Table 2, the gelatinization enthalpy (ΔH) of the branched starch obtained by dual enzyme modification was significant, among which the highest decrease was 7.59 J / g in corn starch treated with cyclodextrin glucosyltransferase and then added with glycogen branching enzyme.

[0079] Table 2 Thermodynamic properties of Examples 1-9

[0080]

[0081] Analysis of the proportion of α-1,6 glycosidic bonds:

[0082] like Figure 5 As shown in Table 3, the α-1,6 glycosidic bond ratio of branched starch modified by dual enzymes is greatly increased compared with native starch, among which the α-1,6 bond ratio of CS-C / G is 19.35%, which is 4.34 times more than that of natural corn starch. The α-1,6 bond ratio of PS-C / G and PBS-C / G are 2.36 times and 2.82 times more than that of natural starch, respectively.

[0083] Table 3 Glycosidic bond ratios of Examples 1-9

[0084]

[0085] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing branched starch, characterized in that: The steps include: (1) dispersing starch in a buffer to obtain starch milk, and preheating the milk at 50-60° C.; (2) adding cyclodextrin glucosyltransferase and glycogen branching enzyme to carry out enzymatic hydrolysis reaction; (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated, the starch is centrifuged, washed, dried, ground and sieved to obtain branched starch.

2. The method for preparing branched starch according to claim 1, characterized in that: In step (1), the mass concentration of starch milk is 10-20%; and the buffer solution is 0.05 mol / L sodium citrate buffer solution.

3. The method for preparing branched starch according to claim 1, characterized in that: In step (1), the starch is a starch having an amylose content greater than 1%, and the starch is one or more of corn starch, potato starch, and pea starch.

4. The method for preparing branched starch according to claim 1, characterized in that: In step (2), the glycogen branching enzyme is from Pyrococcus horikoshii OT3 (Ph), and the cyclodextrin glucosyltransferase is from Bacillus licheniformis.

5. The method for preparing branched starch according to claim 1, characterized in that: In step (2), the added amount of cyclodextrin glucosyltransferase is 0.05-0.15 mL / g dry starch; the added amount of glycogen branching enzyme is 100-500 U / g dry starch.

6. The method for preparing branched starch according to claim 1, characterized in that: In step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added to carry out enzymatic hydrolysis reaction, which specifically includes: after adding cyclodextrin glucosyltransferase for reaction, adjusting the pH to 11-12, standing to inactivate the enzyme, then adjusting the pH to 5-7, and finally adding glycogen branching enzyme for reaction.

7. The method for preparing branched starch according to claim 1, characterized in that: In step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added to carry out enzymatic hydrolysis reaction, which specifically includes: after adding glycogen branching enzyme reaction, adjusting the pH to 11-12, standing to inactivate the enzyme, then adjusting the pH to 5-7, and then adding cyclodextrin glucosyltransferase to react.

8. The method for preparing branched starch according to claim 1, characterized in that: In step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added to carry out enzymatic hydrolysis reaction, which specifically includes: adding cyclodextrin glucosyltransferase and glycogen branching enzyme at the same time to carry out reaction.

9. Branched starch prepared by the method according to any one of claims 1 to 8.

10. Use of the branched starch according to claim 9 in the food industry.

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